Changes In DNA Hydroxymethylation Research Articles

Epigenetic biomarkers of cold and heat syndromes of rheumatoid arthritis: Combining DNA hydroxymethylation and mRNA-sequencing.

Epigenetic biomarkers of cold and heat syndromes of rheumatoid arthritis: Combining DNA hydroxymethylation and mRNA-sequencing.

Transcriptomic and epigenomic analyses uncovered Lrrc15 as a contributing factor to cartilage damage in osteoarthritis

In osteoarthritis (OA), articular chondrocytes display phenotypic and functional changes associated with epigenomic alterations. These changes contribute to the disease progression, which is characterized by dysregulated reparative processes and abnormal extracellular matrix remodeling leading to cartilage degradation. Recent studies using a murine model of posttraumatic OA highlighted the contribution of changes in DNA hydroxymethylation (5hmC) to OA progression. Here, we integrated transcriptomic and epigenomic analyses in cartilage after induction of OA to show that the structural progression of OA is accompanied by early transcriptomic and pronounced DNA methylation (5mC) changes in chondrocytes. These changes accumulate over time and are associated with recapitulation of developmental processes, including cartilage development, chondrocyte hypertrophy, and ossification. Our integrative analyses also uncovered that Lrrc15 is differentially methylated and expressed in OA cartilage, and that it may contribute to the functional and phenotypic alterations of chondrocytes, likely coordinating stress responses and dysregulated extracellular matrix remodeling.

Non-alcoholic fatty liver disease (NAFLD) is associated with dynamic changes in DNA hydroxymethylation

ABSTRACTNon-alcoholic fatty liver disease (NAFLD) is now the commonest cause of liver disease in developed countries affecting 25–33% of the general population and up to 75% of those with obesity. Recent data suggest that alterations in DNA methylation may be related to NAFLD pathogenesis and progression and we have previously shown that dynamic changes in the cell lineage identifier 5-hydroxymethylcytosine (5hmC) may be important in the pathogenesis of liver disease. We used a model of diet-induced obesity, maintaining male mice on a high-fat diet (HFD) to generate hepatic steatosis. We profiled hepatic gene expression, global and locus-specific 5hmC and additionally investigated the effects of weight loss on the phenotype. HFD led to increased weight gain, fasting hyperglycaemia, glucose intolerance, insulin resistance and hepatic periportal macrovesicular steatosis. Diet-induced hepatic steatosis associated with reversible 5hmC changes at a discrete number of functionally important genes. We propose that 5hmC profiles are a useful signature of gene transcription and a marker of cell state in NAFLD and suggest that 5hmC profiles hold potential as a biomarker of abnormal liver physiology.

Changes in DNA hydroxymethylation for the detection of multiple cancers in plasma cell-free DNA.

3058 Background: Methylation and hydroxymethylation of cytosines enable the epigenomic regulation of gene suppression and activation. 5-hydroxymethyl-cytosine (5hmC) is globally decreased in tumor tissue. However, genome-wide analysis using precise 5hmC labelling techniques reveals more nuanced changes upon tumorigenesis and raises the possibility that this loss could be exploited for developing a cancer biomarker. This suggests that 5hmC profiles might enable discrete classification of not only tumor tissue but also of tumor cell-free DNA (cfDNA). We sought to identify genome-wide 5hmC changes in plasma based cfDNA from cancer patients representing multiple disease types, stages and clinical characteristics in comparison with non-cancer patients. Methods: cfDNA was isolated from plasma, enriched for the 5hmC fraction using chemical labelling, sequenced, and aligned to the genome to determine 5hmC counts per genomic feature. Regularized regression models were constructed to classify cancer samples (age matched or corrected for smoking status) on non-overlapping training (80% of all samples) and test sample sets (20% of all samples). Results: 226 non-cancer patients and 278 cancers across four cancer types (breast, colorectal, lung-squamous and pancreas) were included in this study, where more than 60% of cancer samples were early stage disease (I or II). Upon comparison with non-cancer samples, 5hmC peaks have reduced enrichment in exons in breast, colorectal and lung cancer but not in pancreatic cancer. Further, 5hmC peaks in pancreas show different patterns of enrichment in 3’UTR, translational termination sites, promoters and LTR. Overall 5hmC signal density was reduced in late stage cancers across all four diseases. The ability to classify non-cancer versus cancer patients was evaluated via cross-validation of out of fold prediction in the training set with AUC > 0.84 for all four cancer types. Further, test set sensitivity across all four cancer types was found to be > 66% with 98% specificity. Conclusions: These findings suggest that 5hmC changes in plasma cfDNA enable classification of early stages of breast, colorectal, lung-squamous and pancreas cancer and are promising biomarkers for disease detection.

DNA methylation and hydroxymethylation patterns in acute myeloid leukemia patients with mutations in DNMT3A and IDH1/2 and their combinations.

Aberrant epigenetic patterns are a hallmark of acute myeloid leukemia (AML). Mutations in profound epigenetic regulators DNMT3A and IDH1/2 often occur concurrently in AML. The aim was to analyze DNA methylation, hydroxymethylation and mRNA expression profiles in AML with mutations in DNMT3A and IDH1/2 (individually and in combinations). Infinium MethylationEPIC BeadChip (Illumina) covering 850,000 CpGs was utilized. The validation of hydroxy-/methylation data was done by pyrosequencing. HumanHT-12 v4 Expression BeadChip (Illumina) was used for expression examination. Hierarchical clustering analysis of DNA hydroxy-/methylation data revealed clusters corresponding to DNMT3A and IDH1/2 mutations and CD34+ healthy controls. Samples with concurrent presence of DNMT3A and IDH1/2 mutations displayed mixed DNA hydroxy-/methylation profile with preferential clustering to healthy controls. Numbers and levels of DNA hydroxymethylation were low. Uniformly hypermethylated loci in AML patients with IDH1/2 mutations were enriched for immune response and apoptosis related genes, among which hypermethylation of granzyme B (GZMB) was found to be associated with inferior overall survival of AML patients (P= 0.035). Distinct molecular background results in specific DNA hydroxy-/methylation profiles in AML. Site-specific DNA hydroxymethylation changes are much less frequent in AML pathogenesis compared to DNA methylation. Methylation levels of enhancer located upstream GZMB gene might contribute to AML prognostication models.

X-ray irradiation induces subtle changes in the genome-wide distribution of DNA hydroxymethylation with opposing trends in genic and intergenic regions

ABSTRACTDNA hydroxymethylation has gained attention as an intermediate in the process of DNA demethylation. More recently, 5-hydroxymethylcytosine has been recognized as an independent epigenetic mark that can persist over time and that exerts influence on gene regulation and other biological processes. Deregulation of this DNA modification has been linked to tumorigenesis and a variety of other diseases. The impact of irradiation on DNA hydroxymethylation is poorly understood. In this study we exposed lung fibroblasts (IMR90) to 0.5 Gy and 2 Gy of X-rays, respectively. We characterized radiation induced changes of DNA hydroxymethylation 1 h, 6 h, 24 h and 120 h after exposure employing immunoprecipitation and subsequent deep sequencing of the genomic fraction enriched for hydroxymethylated DNA. Transcriptomic response to irradiation was analyzed for time points 6 h and 24 h post exposure by means of RNA sequencing. Irradiated and sham-irradiated samples shared the same overall distribution of 5-hydroxymethylcytosines with respect to genomic features such as promoters and exons. The frequency of 5-hydroxymethylcytosine peaks differentially detected in irradiated samples increased in genic regions over time, while the opposing trend was observed for intergenic regions. Onset and extent of this effect was dose dependent. Moreover, we demonstrated a biased distribution of 5-hmC alterations at CpG islands and sites occupied by the DNA binding protein CTCF. In summary, our study provides new insights into the epigenetic response to irradiation. Our data highlight genomic features more prone to irradiation induced changes of DNA hydroxymethylation, which might impact early and late onset effects of irradiation.

High Dietary Fat Intake Affects DNA Methylation/Hydroxymethylation in Mouse Heart: Epigenetic Hints for Obesity-Related Cardiac Dysfunction.

Epigenetic aberrations caused by environmental factors and lifestyle choices have been associated with the development of a number of pathologies, including cardiovascular disorders. However, whether obesity-related heart dysfunction can occur via epigenetic mechanisms is largely undisclosed. The manifested role of DNA hydroxymethylation in heart pathophysiology prompts an investigation of its levels/machinery in heart of mice fed with high-fat diet (HFD) and its possible relation with genes linked to obesity-associated cardiac remodeling. Alterations in levels of DNA methylation/hydroxymethylation modifications and in expression of Tet family of DNA hydroxylases are observed in hearts of mice treated with HFD for 8 and 16 weeks. Decreased levels of the Tet co-substrate α-ketoglutarate are also observed and associate with mitochondrial mass reduction and augmented oxidative stress. Finally, expression markers of cardiac remodeling are monitored by RT-qPCR analysis and associate with DNA hydroxymethylation signature by DNA immunoprecipitation and correlation analyses. Global changes of DNA hydroxymethylation in hearts of HFD-fed mice are associated with upregulation of the dioxygenase Tet3 and decreased content of α-ketoglutarate. A relation between Tet genes and markers of cardiac hypertrophic response is observed and, if further validated, it will provide insights concerning epigenetics and obesity-related cardiac complications.

DNA hydroxymethylation changes in response to spinal cord damage in a multiple sclerosis mouse model.

Roles of DNA 5-hydroxymethylcytosine (5hmC) in myelin repair were investigated in an experimental autoimmune encephalomyelitis (EAE) mouse model via its regulation on BDNF. DNA 5hmC level and its limiting enzymes were detected in EAE mice. Global 5hmC modification, Tet1 and Tet2 significantly decreased in the spinal cord tissues of EAE mice. BDNF protein and mRNA decreased and were highly associated with BDNF 5hmC. Vitamin C, a Tet co-factor, increased global DNA 5hmC and reduced the neurological deficits at least by increasing BDNF5hmC modification and protein levels. Tet protein-mediated 5hmC modifications represent a critical target involved in EAE-induced myelin damage. Targeting epigenetic modification may be a therapeutic strategy for multiple sclerosis.

Dynamic Transcriptome, DNA Methylome, and DNA Hydroxymethylome Networks During T-Cell Lineage Commitment.

The stepwise development of T cells from a multipotent precursor is guided by diverse mechanisms, including interactions among lineage-specific transcription factors (TFs) and epigenetic changes, such as DNA methylation and hydroxymethylation, which play crucial roles in mammalian development and lineage commitment. To elucidate the transcriptional networks and epigenetic mechanisms underlying T-cell lineage commitment, we investigated genome-wide changes in gene expression, DNA methylation and hydroxymethylation among populations representing five successive stages of T-cell development (DN3, DN4, DP, CD4+, and CD8+) by performing RNA-seq, MBD-seq and hMeDIP-seq, respectively. The most significant changes in the transcriptomes and epigenomes occurred during the DN4 to DP transition. During the DP stage, many genes involved in chromatin modification were up-regulated and exhibited dramatic changes in DNA hydroxymethylation. We also observed 436 alternative splicing events, and approximately 57% (252) of these events occurred during the DP stage. Many stage-specific, differentially methylated regions were observed near the stage-specific, differentially expressed genes. The dynamic changes in DNA methylation and hydroxymethylation were associated with the recruitment of stage-specific TFs. We elucidated interactive networks comprising TFs, chromatin modifiers, and DNA methylation and hope that this study provides a framework for the understanding of the molecular networks underlying T-cell lineage commitment.

Glucose and glutamine availability regulate HepG2 transcriptional responses to low oxygen

Background: Little is known about the impact of nutrients on cellular transcriptional responses, especially in face of environmental stressors such as oxygen deprivation. Hypoxia-inducible factors (HIF) coordinate the expression of genes essential for adaptation to oxygen-deprived environments. A second family of oxygen-sensing genes known as the alpha-ketoglutarate-dependent dioxygenases are also implicated in oxygen homeostasis and epigenetic regulation. The relationship between nutritional status and cellular response to hypoxia is understudied. In vitro cell culture systems frequently propagate cells in media that contains excess nutrients, and this may directly influence transcriptional response in hypoxia. Methods: We studied the effect of glucose and glutamine concentration on HepG2 hepatoma transcriptional response to low oxygen and expression of hypoxia inducible factor-1α (HIF-1α). Mass spectrometry confirmed low oxygen perturbation of dioxygenase transcripts resulted in changes in DNA methylation. Results: Under normoxic conditions, we observed a significant upregulation of both HIF-target genes and oxygen-dependent dioxygenases in HepG2 cells cultured with physiological levels of glucose or glutamine relative to regular DMEM media, demonstrating that excess glutamine/glucose can mask changes in gene expression. Under hypoxic conditions, CA9 was the most upregulated gene in physiological glutamine media while TETs and FTO dioxygenases were downregulated in physiological glucose. Hypoxic regulation of these transcripts did not associate with changes in HIF-1α protein expression. Downregulation of TETs suggests a potential for epigenetic modulation. Mass-spectrometry quantification of modified DNA bases confirmed our transcript data. Hypoxia resulted in decreased DNA hydroxymethylation, which correlated with TETs downregulation. Additionally, we observed that TET2 expression was significantly downregulated in patients with hepatocellular carcinoma, suggesting that tumour hypoxia may deregulate TET2 expression resulting in global changes in DNA hydroxymethylation. Conclusion: Given the dramatic effects of nutrient availability on gene expression, future in vitro experiments should be aware of how excess levels of glutamine and glucose may perturb transcriptional responses.

Glucose and glutamine availability regulate HepG2 transcriptional responses to low oxygen.

Background: Little is known about the impact of nutrients on cellular transcriptional responses, especially in face of environmental stressors such as oxygen deprivation. Hypoxia-inducible factors (HIF) coordinate the expression of genes essential for adaptation to oxygen-deprived environments. A second family of oxygen-sensing genes known as the alpha-ketoglutarate-dependent dioxygenases are also implicated in oxygen homeostasis and epigenetic regulation. The relationship between nutritional status and cellular response to hypoxia is understudied. In vitro cell culture systems frequently propagate cells in media that contains excess nutrients, and this may directly influence transcriptional response in hypoxia. Methods: We studied the effect of glucose and glutamine concentration on HepG2 hepatoma transcriptional response to low oxygen and expression of hypoxia inducible factor-1α (HIF-1α). Mass spectrometry confirmed low oxygen perturbation of dioxygenase transcripts resulted in changes in DNA methylation. Results: Under normoxic conditions, we observed a significant upregulation of both HIF-target genes and oxygen-dependent dioxygenases in HepG2 cells cultured with physiological levels of glucose or glutamine relative to regular DMEM media, demonstrating that excess glutamine/glucose can mask changes in gene expression. Under hypoxic conditions, CA9 was the most upregulated gene in physiological glutamine media while TETs and FTO dioxygenases were downregulated in physiological glucose. Hypoxic regulation of these transcripts did not associate with changes in HIF-1α protein expression. Downregulation of TETs suggests a potential for epigenetic modulation. Mass-spectrometry quantification of modified DNA bases confirmed our transcript data. Hypoxia resulted in decreased DNA hydroxymethylation, which correlated with TETs downregulation. Additionally, we observed that TET2 expression was significantly downregulated in patients with hepatocellular carcinoma, suggesting that tumour hypoxia may deregulate TET2 expression resulting in global changes in DNA hydroxymethylation. Conclusion: Given the dramatic effects of nutrient availability on gene expression, future in vitro experiments should be aware of how excess levels of glutamine and glucose may perturb transcriptional responses.

Association of high 5-hydroxymethylcytosine levels with Ten Eleven Translocation 2 overexpression and inflammation in Sjögren's syndrome patients

Here, we determined the 5-hydroxymethylcytosine (5hmC), 5-methylcytosine (5mC), Ten Eleven Translocation (TETs), and DNA methyltransferases (DNMTs) levels in epithelial and inflammatory cells of labial salivary glands (LSG) from Sjögren's syndrome (SS)-patients and the effect of cytokines on HSG cells. LSG from SS-patients, controls and HSG cells incubated with cytokines were analysed. Levels of 5mC, 5hmC, DNMTs, TET2 and MeCP2 were assessed by immunofluorescence. In epithelial cells from SS-patients, an increase in TET2, 5hmC and a decrease in 5mC and MeCP2 were observed, additionally, high levels of 5mC and DNMTs and low levels of 5hmC were detected in inflammatory cells. Cytokines increased TET2 and 5hmC and decreased 5mC levels. Considering that the TET2 gene.promoter contains response elements for transcription factors activated by cytokines, together to in vitro results suggest that changes in DNA hydroxymethylation, resulting from altered levels of TET2 are likely to be relevant in the Sjögren's syndrome etiopathogenesis.

Hypoxia Drives Breast Tumor Malignancy through a TET-TNFα-p38-MAPK Signaling Axis.

Hypoxia is a hallmark of solid tumors that drives malignant progression by altering epigenetic controls. In breast tumors, aberrant DNA methylation is a prevalent epigenetic feature associated with increased risk of metastasis and poor prognosis. However, the mechanism by which hypoxia alters DNA methylation or other epigenetic controls that promote breast malignancy remains poorly understood. We discovered that hypoxia deregulates TET1 and TET3, the enzymes that catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby leading to breast tumor-initiating cell (BTIC) properties. TET1/3 and 5hmC levels were closely associated with tumor hypoxia, tumor malignancy, and poor prognosis in breast cancer patients. Mechanistic investigations showed that hypoxia leads to genome-wide changes in DNA hydroxymethylation associated with upregulation of TNFα expression and activation of its downstream p38-MAPK effector pathway. Coordinate functions of TET1 and TET3 were also required to activate TNFα-p38-MAPK signaling as a response to hypoxia. Our results reveal how signal transduction through the TET-TNFα-p38-MAPK signaling axis is required for the acquisition of BTIC characteristics and tumorigenicity in vitro and in vivo, with potential implications for how to eradicate BTIC as a therapeutic strategy.

Hydroxymethylation and DNA methylation profiles in the prefrontal cortex of the non-human primate rhesus macaque and the impact of maternal deprivation on hydroxymethylation

5-Hydroxymethylcytosine (5hmC) is abundant in the brain, suggesting an important role in epigenetic control of neuronal functions. In this paper, we show that 5hmC and 5-methylcytosine (5mC) levels are coordinately distributed in gene promoters of the rhesus macaque prefrontal cortex. Although promoter hydroxymethylation and methylation are overall negatively correlated with expression, a subset of highly expressed genes involved in specific cerebral functions is associated with high levels of 5mC and 5hmC. These relationships were also observed in the mouse cortex. Furthermore, we found that early-life maternal deprivation is associated, in the adult monkey cortex, with DNA hydroxymethylation changes of promoters of genes related to neurological functions and psychological disorders. These results reveal that early social adversity triggers variations in brain DNA hydroxymethylation that could be detected in adulthood.

Aging alters global hepatic DNA hydroxymethylation in mice, as determined by a novel LC/MS‐MS method

Aging is associated with changes in global DNA methylation (mC), but changes in DNA hydroxymethylation (hmC) are not established due to a relative lack of methods available to measure hmC. Both modifications can affect gene expression and may be important in aging. We have developed a method to measure 5′‐methyl‐2′‐deoxycytidine and 5′‐hydroxymethyl‐2′‐deoxycytidine in DNA using liquid chromatography/tandem mass spectrometry (LC/MS‐MS) and used this method to assess global hmC and mC levels in hepatic tissue from young (9 months; n=10) and old (23 months; n=6) C57BL/6 male mice. DNA was enzymatically hydrolyzed and isotopomers [15N3]‐2′‐deoxycytidine and (methyl‐d3, ring‐6‐d1)‐5‐methyl‐2′‐deoxycytidine were added as internal standards. The mixture of DNA hydrolysates and internal standards were separated through LC, and nucleosides of interest were detected by MS‐MS in positive ion mode using multiple reaction monitoring. Global mC and hmC levels were calculated as a percentage of total deoxycytidine residues in genomic DNA. Liver tissue from old mice had significantly higher global hmC relative to young mice (0.32±0.02% vs 0.24±0.01%; p=0.02), while mC levels did not change. Using this new method we have found that aging is associated with an increase in global DNA hmC, a finding that may be useful in understanding the physiological mechanisms behind aging.Grant Funding Source: NIH grant R01 AG025834

Genome-wide DNA hydroxymethylation changes are associated with neurodevelopmental genes in the developing human cerebellum

5-Hydroxymethylcytosine (5-hmC) is a newly discovered modified form of cytosine that has been suspected to be an important epigenetic modification in neurodevelopment. While DNA methylation dynamics have already been implicated during neurodevelopment, little is known about hydroxymethylation in this process. Here, we report DNA hydroxymethylation dynamics during cerebellum development in the human brain. Overall, we find a positive correlation between 5-hmC levels and cerebellum development. Genome-wide profiling reveals that 5-hmC is highly enriched on specific gene regions including exons and especially the untranslated regions (UTRs), but it is depleted on introns and intergenic regions. Furthermore, we have identified fetus-specific and adult-specific differentially hydroxymethylated regions (DhMRs), most of which overlap with genes and CpG island shores. Surprisingly, during development, DhMRs are highly enriched in genes encoding mRNAs that can be regulated by fragile X mental retardation protein (FMRP), some of which are disrupted in autism, as well as in many known autism genes. Our results suggest that 5-hmC-mediated epigenetic regulation may broadly impact the development of the human brain, and its dysregulation could contribute to the molecular pathogenesis of neurodevelopmental disorders. Accession number: Sequencing data have been deposited to GEO with accession number GSE40539.